Collagenase Activity Research Articles

In silico analysis of Arbacia lixula-derived peptides and plasmid construction for recombinant anti-aging therapies

Skin aging is one of the degenerative processes influenced by tyrosinase, elastase, collagenase, hyaluronidase, and matrix metalloproteinase-9 (MMP9) activity. One promising avenue for discovering antiaging therapeutics is the peptides from the Arbacia lixula spine. The aim of this study was to explore the potential of peptides from A. lixula spine as a multitarget inhibitor for recombinant antiaging therapies through in silico approaches. The crystal structure of peptides previously identified in A. lixula spine was visualized using the UCSF Chimera. The protein data bank (PDB) database was used to obtain the crystal structures of protein targets. The webservers Innovagen, AllerTop, and ToxinPred were utilized to predict the peptide's water solubility, toxicity, and allergenicity. MOE application was used to prepare all ligands and proteins, molecular docking, and visualization. Molecular dynamics simulations were carried out on the protein-ligand complexes on Yasara Dynamics application. The Benchling website was used to perform virtual electrophoresis and reconstruct the recombinant plasmid (Psb1c3). Based on the molecular docking results, peptide REGSPDLLE has the potential as a multitarget inhibitor of tyrosinase (-9.07 kcal/mol), hyaluronidase (-10.57 kcal/mol), elastase (-9.32 kcal/mol), collagenase (-10.57 kcal/mol), and MMP9 (-10.43 kcal/mol). Peptide REGSPDLLE was selected due to its strong binding affinity on the active site of each target protein and exhibits non-toxic, non-allergenic, and good water-soluble as indicated by Support Vector Machine score <0. Molecular dynamics simulations confirmed stable interactions with receptor proteins. Peptide REGSPDLLE was successfully inserted into the recombinant pSB1C3 plasmid, confirmed by virtual electrophoresis with bands at ~2000 bp and ~150 bp. Further in vitro and in vivo studies are necessary to verify the anti-aging efficacy of peptide REGSPDLLE.

Scaling up the optimized production of Aspergillus heteromorphus URM0269 collagenase in soybean agroindustrial residue

Collagenase and protease productions from Aspergillus heteromorphus URM0269 were optimized by submerged fermentation using soybean flour as substrate. Fermentations were performed according to composite design using the concentrations of substrate and yeast extract as the independent variables. The best condition was scaled up in a stirred tank bioreactor to assess the fermentation kinetics. The highest production of both enzymes occurred at concentrations of 2.0 % substrate and 0.1 % yeast extract. Contrariwise, after scale-up, collagenase activity increased from 33.5 to 148.5 U/mL, while the protease decreased from 16.3 to 11.7 U/mL. A. heteromorphus URM0269 showed a maximum growth rate of 0.09 h−1 and yields of protease and collagenase on biomass, after 65 h of 2.70 and 34.22 U/mgx, respectively. Collagenase acted optimally at 40 °C and pH 6.0 on collagen as a substrate and displayed an allosteric trend, achieving a maximum reaction rate of 132.47 U/mL. Thermodynamic parameters of collagen degradation such as Gibbs free energy (74.16 kJ/mol), enthalpy (11.64 kJ/mol), entropy (−199.63 J/K.mol), and activation energy (14.25 kJ/mol) were determined for optimal temperature. These results demonstrated that soybean flour is a potential agroindustrial residue for collagenase production. Furthermore, the collagenase displayed promising biochemical and thermodynamic features for other biotechnological applications.

What Is the Role of the Gut Microbiota in Anastomotic Leakage After Colorectal Resection? A Scoping Review of Clinical and Experimental Studies.

Background: Anastomotic leakage (AL) still remains a common complication after colorectal anastomosis that leads to increased morbidity and mortality. The gut microbiota has been hypothesized as one of the risk factors associated with anastomotic leakage. The aim of the present study was to summarize all existing clinical and experimental studies that evaluate the impact of intestinal microbiota on anastomotic leakage after colorectal resection. Methods: The present scoping review was designed according to PRISMA recommendations and a systematic search in Medline, Scopus, EMBASE, Clinicaltrials.gov, Google Scholar, and CENTRAL was conducted until September 2024. Results: Overall, 7 clinical and 5 experimental studies were included. A diminished α-diversity of the gut microbiota in patients suffering from AL was demonstrated. Specific microbe genera, such as Lachnospiraceae, Bacteroidaceae, Bifidobacterium, Acinetobacter, Fusobacterium, Dielma, Elusimicronium, Prevotella, and Faecalibacterium, seem to be associated with AL. However, specific genera, like Prevotella, Streptococcus, Eubacterium, Enterobacteriaceae, Klebsiella, Actinobacteria, Gordonibacter, Phocaeicola, and Ruminococcus2, seem to be protective against AL. Experimental studies highlighted that the Western diet seems to affect microbiota diversity and increases the AL rate, whereas anastomotic healing seems to be impaired by high metalloproteinase production and increased collagenase activity. Conclusions: The intestinal microbiota seems to play an important role in anastomotic leakage after colorectal resection. Specific interventions targeting the microbiota's composition and the pathophysiological mechanisms by which it impairs anastomotic healing could diminish the risk for anastomotic leakage and improve clinical outcomes. However, future studies should be based on prospective design and eliminate heterogeneity.

Characterization and anti-aging activities of Haruan (Channa striata) fish oil

Haruan fish or snakehead fish (Channa striata) is a freshwater fish widely cultivated in Indonesia. Fish oils are believed to have some biological activities which are beneficial to human health, including antioxidant and antiaging activities. The objectives of this study are to determine the characteristics and yield of Haruan fish oil (HFO) using three extraction methods (steaming, maceration, and pressing), to determine radical scavenging activity (RSA) of fish oils using 2,2’-diphenyl-1-picrylhydrazyl (DPPH) radicals and 2,2'- azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals and to determine anticollagenase activity of HFO. The extraction of HFO from the flesh part using the steaming method resulted in the highest RSA values either using DPPH or ABTS radicals. HFO could inhibit the activity of collagenase by 26.87±0.391%. The highest yield of HFO was obtained using the steaming method, with extraction yields of flesh and offal of 2.02% and 2.05%, respectively. The acid values (AV) of HFOs obtained from different parts using the steaming method were 1.87±0.04 (flesh part), 1.88±0.07 (head) and 1.93±0.05 (offal). The saponification values (SV) of HFO using the same extraction method were 6.30±0.26, 6.17±0.07, and 6.30±0.23 for flesh, head and offal, respectively. The peroxide (PV) and iodine (IV) values were 85.37±2.86 and 198.51±7.68, 103.70±2.21 and 206.44±5.24, as well as 96.05±2.93 and 193.68±1.70, for flesh, head and offal, respectively. These parameters (AV, SV, PV and IV) met the requirements. Haruan fish oil has the potential to be developed as a source of the human diet.

Modulation of the K/BxN arthritis mouse model and the effector functions of human fibroblast-like synoviocytes by liver X receptors.

The role of liver X receptors (LXR) in rheumatoid arthritis (RA) remains controversial. We studied the effect of LXR agonists on fibroblast-like synoviocytes (FLS) from RA patients and the K/BxN arthritis model in LXRα and β double-deficient (Nr1h2/3-/-) mice. Two synthetic LXR agonists, GW3965 and T0901317, were used to activate LXRs and investigate their effects on cell growth, proliferation and matrix metalloproteinases, and chemokine production in cultured FLS from RA patients. The murine model K/BxN serum transfer of inflammatory arthritis in Nr1h2/3-/- animals was used to investigate the role of LXRs on joint inflammation in vivo. LXR agonists inhibited the FLS proliferative capacity in response to TNF, the chemokine-induced migration, the collagenase activity in FLS supernatant and FLS CXCL12 production. In the K/BxN mouse model, Nr1h2/3-/- animals showed aggravated arthritis, histological inflammation, and joint destruction, as well as an increase in synovial metalloproteases and expression of proinflammatory mediators such as IL-1β and CCL2 in joints compared with wild type animals. Taken together, these data underscore the importance of LXRs in modulating the joint inflammatory response and highlight them as potential therapeutic targets in RA.

A Piper nigrum based zinc oxide nanoparticles for anti-arthritic and antioxidant activity

Background: Zinc oxide nanoparticles (ZnONPs) are among the most effective metallic oxide nanoparticles for biological applications. They have potential anti-inflammatory and anti-oxidant properties, desirable biocompatibility, lower toxicity, and minimal cost. Methodology: Using diverse plant extracts for an ecologically friendly production of metallic nanoparticles is a better choice than conventional chemical synthesis techniques. The present study is decisive on the ZnONPs synthesis from a Piper nigrum extract (Pn-ZnONPs). Morphological characteristics of ZnONPs have been studied using UV-spectroscopy, DLS, SEM, and TEM. Further, it is analyzed for its anti-inflammatory (proteinase, collagenase, lipooxygenase, and elastase) and anti-oxidant properties (DPPH˙, SOD, NO, H2O2, and OH). Results and discussion: Synthesis of nanoparticles has been confirmed via visible spectroscopy with maximum absorbance of 350nm, having particle size and zeta potential of 80 nm and +7.4 mV, respectively. SEM and TEM analysis confirmed the nanoparticle's shape to be spherical and arranged compactly. Further, biogenic nanoparticles show desired anti-inflammatory properties by inhibiting the activity of collagenase (68.72%), elastase (65.16%), lipooxygenase (58.098%), and denaturation of protein (65.36%). It also exhibits the capability to suppress Superoxide radicals (64.87%), DPPH (65.46%), Hydrogen Peroxide (64.89%), Hydroxyl radical (68.45%), and Nitric oxide radicals (71.343%), which are responsible for the pathogenesis of many inflammatory disorders. Conclusion: This suggests that P. nigrum Zinc nanoparticles may be a promising agent in treating various inflammatory disorders, such as cancer, Rheumatoid Arthritis, Psoriasis, and others.

High keratinase and other types of hydrolase activity of the new strain of Bacillus paralicheniformis.

Keratinases, a subclass of proteases, are used to degrade keratin thereby forming peptones and free amino acids. Bacillus paralicheniformis strain T7 was isolated from soil and exhibited high keratinase, protease, collagenase, amylase, xylanase, lipase, and phosphatase activities. Keratinases of the strain showed maximum activity at 70°C and pH 9.0 as well as high thermal stability. A mass-spectrometric analysis identified seven peptidases with molecular masses of 26.8-154.8 kDa in the secretory proteome. These peptidases are members of S8 and S41 serine peptidase families and of M14, M42, and M55 metallopeptidase families. Additionally, α-amylase (55.2 kDa), alkaline phosphatase (59.8 kDa), and esterase (26.8 kDa) were detected. The strong keratinolytic properties of the strain were confirmed by degradation of chicken and goose feathers, which got completely hydrolyzed within 4 days. Submerged fermentation by strain B. paralicheniformis T7 was carried out in a pilot bioreactor, where the highest keratinase production was noted after 19 h of cultivation. After the fermentation, in the culture fluid, the keratinase activity toward keratin azure was 63.6 ± 5.8 U/mL. The protease activity against azocasein was 715.7 ± 40.2 U/mL. The possibility of obtaining enzyme preparations in liquid and powder form was demonstrated, and their comparative characteristics are given. In the concentrate, the keratinase, protease, α-amylase, phosphatase, and esterase/lipase activities were 2,656.7 ± 170.4, 29,886.7 ± 642.9, 176.1 ± 16.3, 23.9 ± 1.8, and 510.9 ± 12.2 U/mL, respectively. In the lyophilizate, these activities were 57,733.3 ± 8,911.4, 567,066.7 ± 4,822.2, 2,823.0 ± 266.8, 364.2 ± 74.8, and 17,618.0 ± 610.3 U/g, respectively. In the preparation obtained by air flow drying at 55°C, these activities were 53,466.7 ± 757.2, 585,333.3 ± 4,277.1, 2,395.8 ± 893.7, 416.7 ± 52.4, and 15,328.1 ± 528.6 U/g, respectively. The results show high potential of B. paralicheniformis strain T7 as a producer of keratinases and other enzymes for applications in agricultural raw materials and technologies for processing of keratin-containing animal waste.

THE INFLUENCE OF THE CULTURE OF FIBROBLASTIC CELL ELEMENTS ON THE INDICATORS OF THE METABOLISM OF CONNECTIVE TISSUE IN EXPERIMENTAL ANIMALS

The number of patients with degenerative tendon disease affects millions of people both among athletes and the general population, causing significant socio-economic consequences. Despite the availability of various methods of conservative and surgical treatment, more than a third of patients experience constant pain. Objective. To study the indicators of the metabolism of connective tissue in animals with a model of degenerative damage to tendons against the background of the introduction of a culture of fibroblastic cell elements. Methods. Therefore, the development of methods for restoring the structure of tendons using cell cultures, in particular fibroblasts, will allow to optimize the course of reparative processes, reduce the risk of complications during surgical intervention and accelerate healing, and at the molecular level — to improve the structure of collagen fibers. Laboratory studies of biochemical markers of a tendon with a degenerative-dystrophic lesion and against the background of the introduction of cell culture can help in the differential diagnosis of its extracellular matrix. Results. The experimental data obtained by us indicate the presence of differences in the biochemical markers of tendons with degenerative-dystrophic lesions in rats 7, 21, and 45 days after the introduction of culture of fibroblastic cell elements. However, 45 days after the introduction of the culture of fibroblast cell elements, the normalization of metabolic processes in the extracellular matrix of connective tissue occurs, namely, the activity of collagenase and the concentration of protein-bound hydroxyproline approaches normal values. This indicates the predominance of the synthetic phase over the catabolic one in collagen metabolism. Conclusions. In this context, the introduction of culture of fibroblastic cell elements, as an alternative anti-inflammatory method, may provide another potential opportunity in the treatment of chronic degenerative-dystrophic lesions of the Achilles tendon.

Inhibitory Effects of Polyphenols from Equisetum ramosissimum and Moringa peregrina Extracts on Staphylococcus aureus, Collagenase, and Tyrosinase Enzymes: In vitro Studies

Background: Skin problems caused by oxidative stress lead to the activation of collagenase and tyrosinase enzymes, contributing to skin aging, discoloration, and infections. Equisetum ramosissimum and Moringa peregrina were assessed for their potential uses in treating various skin conditions. Objective: The present research aimed to investigate the positive effects of polyphenols in Equisetum ramosissimum and Moringa peregrina extracts as potential cosmetic products for the treatment of different skin conditions. Methods: Total phenolic and flavonoid contents, antioxidants, and anti-collagenase and anti-tyrosinase activities of plant extract mixtures (PEM) at different ratios of (M. peregrina: E. ramosissimum) were determined using standard procedures. Inhibitory effects of PEM against acne-causing Staphylococcus aureus (ATCC 29213) were evaluated using the diameter (cm) of the inhibition zone method. A cream formulation containing PEM was developed and characterized for stability and potential skin irritation in rats using standard procedures. Results: The PEM at a ratio of (2:1) showed the highest total phenolic and flavonoid content (150.15 ± 2.8 mg/g, equivalent to gallic acid, and 41.5 ± 1.2 mg/g, equivalent to quercetin, respectively). Antioxidant activities for PEM (2:1) were also optimal, as determined by the DPPH and ABTS methods (IC50 = 7.06 ± 0.12 µg/mL and 53.29 ± 3.3 µg/mL, respectively). Furthermore, PEM (2:1) exhibited superior inhibitory activities against collagenase and tyrosinase enzymes (IC50 = 32.4 ± 1.19 µg/mL and 8.4 ± 1.19 µg/mL, respectively). Antimicrobial activity of PEM (2:1) tested on S. aureus showed the largest zone of growth inhibition (2.8 cm) at a concentration of 60 mg/mL. Studies on the PEM (2:1) cream formulation revealed that it remained stable under room conditions. Skin irritation tests on rats showed no signs of oedema or erythema after treatment. Conclusion: The PEM with a ratio of (2:1) demonstrated optimal activity as an oxidative stress-neutralizing agent, inhibitor of enzymes responsible for skin aging and hyperpigmentation, and antibacterial agent. The cream formulation containing PEM exhibited physical stability and no detectable risk of skin irritation throughout the research procedures.

Unlocking the Potential of Collagenases: Structures, Functions, and Emerging Therapeutic Horizons.

Collagenases, a class of enzymes that are specifically responsible for collagen degradation, have garnered substantial attention because of their pivotal roles in tissue repair, remodeling, and medical interventions. This comprehensive review investigates the diversity, structures, and mechanisms of collagenases and highlights their therapeutic potential. First, it provides an overview of the biochemical properties of collagen and highlights its importance in extracellular matrix function. Subsequently, it meticulously analyzes the sources of collagenases and their applications in tissue engineering and food processing. Notably, this review emphasizes the predominant role played by microbial collagenases in commercial settings while discussing their production and screening methods. Furthermore, this study elucidates the methodology employed for determining collagenase activity and underscores the importance of an accurate evaluation for both research purposes and clinical applications. Finally, this review highlights the future research prospects for collagenases, with a particular focus on promoting wound healing and treating scar tissue formation and fibrotic diseases.

Towards the Use of Lichens as a Source of Bioactive Substances for Topical Applications.

The increasing incidence of dermatological diseases prompts the search for new natural methods of treatments, and lichens, with their special symbiotic structure, are a little-known and promising source of biologically active substances. Seven lichen species, Cladonia unicialis (L.) Weber ex F.H. Wigg. (Cladoniaceae), Evernia prunastri (L.) Ach. (Parmeliaceae), Hypogymnia physodes (L.) Nyl. (Parmaliaceae), Parmelia sulcata (Taylor) (Parmeliaceae), Physcia adscendens (Fr.) H. Olivier (Physciaceae), Pseudoevernia furfuracea (L.) Zopf (Parmeliaceae), and Xanthoria parietina (L.) Th. Fr. (Teloschistaceae), were used in our experiment. We identified different metabolites in the acetone extracts of all the lichen species. Based on the high-performance liquid chromatography analysis, the content of lichen substances in the extracts was evaluated. The impact of the individual lichen-specific reference substances, compared to the lichen extracts, on the viability of keratinocytes (HaCaT cell line) and fibroblasts (BJ cell line) and on the activity of selected skin-related enzymes was investigated. Our results revealed that only emodin anthrone at a concentration of 200 mg/L was cytotoxic to keratinocytes and fibroblasts in both cell viability assays. In turn, the C. uncialis extract was only cytotoxic to keratinocytes when used at the same concentration. The other tested treatments showed a positive effect on cell viability and no cytotoxicity or indeterminate cytotoxicity (shown in only one of the tests). Elastase and collagenase activities were inhibited by most of the lichen extracts. In turn, the individual lichen compounds (with the exception of evernic acid) generally had an undesirable stimulatory effect on hyaluronidase and collagenase activity. In addition, almost all the tested compounds and extracts showed anti-inflammatory activity. This suggests that some lichen compounds hold promise as potential ingredients in dermatological and skincare products, but their safety and efficacy require further study. The high cytotoxicity of emodin anthrone highlights its potential use in the treatment of hyperproliferative skin diseases such as psoriasis.

Evaluation of Biotechnological Active Peptides Secreted by Saccharomyces cerevisiae with Potential Skin Benefits.

Biotechnological active peptides are gaining interest in the cosmetics industry due to their antimicrobial, anti-inflammatory, antioxidant, and anti-collagenase (ACE) effects, as well as wound healing properties, making them suitable for cosmetic formulations. The antimicrobial activity of peptides (2-10 kDa) secreted by Saccharomyces cerevisiae Ethanol-Red was evaluated against dermal pathogens using broth microdilution and challenge tests. ACE was assessed using a collagenase activity colorimetric assay, antioxidant activity via spectrophotometric monitoring of nitrotetrazolium blue chloride (NBT) reduction, and anti-inflammatory effects by quantifying TNF-α mRNA in lipopolysaccharides (LPS)-exposed dermal fibroblasts. Wound healing assays involved human fibroblasts, endothelial cells, and dermal keratinocytes. The peptides (2-10 kDa) exhibited antimicrobial activity against 10 dermal pathogens, with the Minimum Inhibitory Concentrations (MICs) ranging from 125 µg/mL for Staphylococcus aureus to 1000 µg/mL for Candida albicans and Streptococcus pyogenes. In the challenge test, peptides at their MICs reduced microbial counts significantly, fulfilling ISO 11930:2019 standards, except against Aspergillus brasiliensis. The peptides combined with MicrocareⓇ SB showed synergy, particularly against C. albicans and A. brasilensis. In vitro, the peptides inhibited collagenase activity by 41.8% and 94.5% at 250 and 1000 µg/mL, respectively, and demonstrated antioxidant capacity. Pre-incubation with peptides decreased TNF-α expression in fibroblasts, indicating anti-inflammatory effects. The peptides do not show to promote or inhibit the angiogenesis of endothelial cells, but are able to attenuate fibrosis, scar formation, and chronic inflammation during the final phases of the wound healing process. The peptides showed antimicrobial, antioxidant, ACE, and anti-inflammatory properties, highlighting their potential as multifunctional bioactive ingredients in skincare, warranting further optimization and exploration in cosmetic applications.

Comparative Evaluation of Soft Tissue Reaction to Titanium and Polyetheretherketone Customized Healing Abutments: a Split-Mouth Randomised Clinical Trial

Aim. This study aimed to evaluate the peri-implant soft tissue response to customized healing abutments (CHAs) made of titanium and polyetheretherketone (PEEK). Methods. Titanium and PEEK CHAs were mounted on bone-level dental implants in bilateral edentulous patients in a randomized split-mouth study design. After a three-week healing period, the clinical parameters of the peri-implant soft tissues were assessed. The value of marginal bone resorption was measured by cone-beam computed tomography. In addition, peri-implant crevicular fluid samples were obtained to identify periodontal pathogenic bacteria, cytokine levels, and active neutrophil collagenase (aMMP-8) concentration. Microcirculation and oxygenation parameters were assessed using laser Doppler flowmetry and optical tissue oximetry. In addition, wavelet analysis was conducted to study blood flow control mechanisms. Results. Out of the 23 participants initially recruited, 20 were assessed after titanium and PEEK CHA installation due to losses to follow-up. The peri-implant soft tissue around 27 titanium and 25 PEEK CHAs was evaluated during follow-ups. No differences were found in the values of marginal bone resorption, cytokine levels, or total bacterial load (p>0.05). PEEK CHAs demonstrated lower bleeding on probing values, less plaque accumulation, and faster relief of ischemia and hypoxia. Although aMMP-8 concentration was higher in the PEEK group, the values were within normal limits. Conclusions. The results of this comparative study showed a positive soft tissue response to PEEK CHAs. However, further studies comparing the effectiveness of different materials for CHAs should be carried out, considering their specific physical properties and processing protocols.

MMP13 Expression and Activity Suggest Its Role in Bone Resorption in Ameloblastomas.

Ameloblastoma is a locally destructive benign odontogenic tumor. While the neoplastic cells of conventional ameloblastoma can infiltrate the connective tissue and bone, in unicystic ameloblastoma the epithelium is encapsulated. The mechanisms driving ameloblastoma's bone resorption remains unclear. RNA sequencing (RNA-seq) was performed in a discovery cohort of conventional ameloblastoma, and pathway enrichment analysis was carried out. mRNA levels of MMP13, a gene associated with bone resorption, were assessed using RT-qPCR in a larger cohort of conventional ameloblastoma and in unicystic ameloblastoma. Zymogram gels and the immunoexpression profile of collagenase 3 (encoded by MMP13 gene) were evaluated as well. Enriched pathways related to bone mineralization and upregulation of MMP13 were observed in ameloblastomas. Collagenolytic activity of collagenase 3 was detected in the tumor lysates. Collagenase 3 immunopositivity was observed in ameloblastomatous epithelium infiltrating the fibrous capsule of unicystic ameloblastoma. At the tumor-bone interface, collagenase 3 expression was detected in stromal cells, osteoblasts, and osteocytes. The results indicate a potential involvement of MMP13 in ameloblastoma-related bone resorption and progression.

Investigating the inhibitory effects of carotenoids-fortified Helix aspersa slime on oxidative stress, collagenase and tyrosinase enzyme activities.

The investigations of snail slime and its possible biological activities have been performed recently. This study aimed to evaluate the properties of Helix aspersa snail slime (HAS), and carotenoids-fortified slime (HASC). Snails were fed with carrots for 10 days. Slime was collected and extracted using water. Saponins identification, antioxidant, collagenase, and tyrosinase enzyme inhibition activities, carotenoids, total phenols, and flavonoids content, were determined. UHPLC-MS/MS analysis was performed for phytochemical characterization. Saponins were detected in the HAS extract only. However, HASC was shown to contain a higher carotenoid content than HAS (29.51 ± 0.4 vs. 18.11 ± 0.2 μg/g). similarly, total phenolic and flavonoid content were higher for the the HASC extract compared to the HAS (182.3 ± 5.2 vs. 150.28 ± 3.3 mg/g equivalent to gallic acid), and (77.62 ± 1.2 vs. 14.19 ± 0.9 mg/g equivalent to quercetin). As expected, the HASC extract exhibited higher antioxidant activity compared to the HAS, using DPPH and the ABTS assays (IC50 = 7.75 ± 0.14 vs. 20.1 ± 0.4 μg/mL), and (IC50 = 7.6 ± 0.26 vs. 19.57 ± 1.4 μg/mL). UHPLC-MS/MS analysis revealed the presence of several phytocomponents of which, hexadecanoic acid and ascorbic acid, were observed in the HASC extract. Furthermore, HASC extract exhibited superior inhibitory activity compared to HAS against collagenase and tyrosinase enzymes (IC50 = 8.4 ± 1.19 vs. 15.3 ± 1.12 μg/mL) and (IC50 = 30.1 ± 0.91 vs. 35 ± 1.3 μg/mL). These findings highlight the potential of HASC as a valuable ingredient in various pharmaceutical applications, due to their content of various phenolic, antioxidants, carotenoids, hexadecanoic, and ascorbic acids. The latter is well known for its great cosmeceutical applications used for slowing the process of skin aging.

A near-infrared light-driven Janus nanomotor for deep tumor penetration and enhanced tumor immunotherapy.

A near-infrared light-driven Janus nanomotor is constructed by collagenase-coated gold nanorods and chitosan-functionalized mesoporous organosilica nanoparticles with Mn2+ as the bridging ion. The nanomotors with excellent motility and collagenase activity can potently penetrate into tumors to sufficiently activate innate immune responses, significantly enhancing anti-tumor immune efficacy in vivo.

ESSENTIAL OIL CONTENTS AND BIOLOGICAL ACTIVITIES OF THYMUS CANOVIRIDIS JALAS AND THYMUS SIPYLEUS BOISS.

Objective: Members of the Lamiaceae family are considered to be major sources of bioactive therapeutic agents. Many of them are important medicinal and aromatic plants used in traditional and modern medicine and in the food, cosmetic and pharmaceutical industries. The aim of this study was to investigate in detail the biological activities and chemical composition of the essential oils of Thymus canoviridis Jalas and Thymus sipyleus Boiss. species belonging to the genus Thymus, one of the most important genera of the Lamiaceae family. Material and Method: The essential oil content of the species was determined by GC-MS. Antioxidant activities of the essential oils were determined using lipid peroxidation, DPPH free radical, ABTS cation radical and CUPRAC methods. In addition, cytotoxic activities against breast cancer (MCF-7) and colon cancer (HT-29) cell lines and anticholinesterase (against AChE and BChE enzymes), urease, tyrosinase, elastase, collagenase and angiotensin converting enzyme inhibition activities were determined. Result and Discussion: When the essential oil composition of T. sipyleus was analyzed, the major compounds were 1,8-cineole (eucalyptol) (18.16%), camphor (15.08%) and endo-borneol (11.63%), while T. canoviridis was found to be rich in carvacrol (72.88%). T. canoviridis showed high antioxidant activity in lipid peroxidation (IC50: 45.72±0.12 μg/ml), ABTS (IC50: 6.12±0.03 μg/ml) and CUPRAC (IC50: 5.31±0.01 μg/ml) methods. The selectivities of T. canoviridis and T. sipyleus species against MCF-7 cell line were 4.39 and 6.81, respectively. In the enzyme inhibition studies, both Thymus species showed moderate inhibition activity against BChE enzyme (Inhibition%: 57.88±1.14, 39.21±0.89, respectively). In addition, T. sipyleus showed moderate inhibition of elastase enzyme (Inhibition%: 25.33±0.79). When the results are evaluated in general, it can be said that T. canoviridis essential oil with its rich carvacrol content and high cytotoxic and antioxidant activity can be preferred as a safer and natural option instead of synthetic preservatives in food, pharmaceutical and cosmetic industries to extend shelf life and ensure food safety.

Engineering a dysbiotic biofilm model for testing root caries interventions through microbial modulation

BackgroundThis study aimed to engineer and optimise a dysbiotic biofilm model to develop in vitro root caries for investigating microbial modulation strategies. The model involved growing complex biofilms from a saliva inoculum collected from four volunteers using two strategies. In the first strategy (“pre-treatment strategy”), bovine root slabs were used, and two natural compounds were incorporated at time 0 of the 10-day biofilm experiment, which included sucrose cycles mimicking the cariogenic environment. In the second strategy (“post-treatment strategy”), mature biofilms were grown in a modified Calgary biofilm device coated with collagen and hydroxyapatite for 7 days and then were exposed to the same natural compounds. The metatranscriptome of each biofilm was then determined and analysed. Collagenase activity was examined, and the biofilms and dentine were imaged using confocal and scanning electron microscopy (SEM). Mineral loss and lesion formation were confirmed through micro-computed tomography (μ-CT).ResultsThe pH confirmed the cariogenic condition. In the metatranscriptome, we achieved a biofilm compositional complexity, showing a great diversity of the metabolically active microbiome in both pre- and post-treatment strategies, including reads mapped to microorganisms other than bacteria, such as archaea and viruses. Carbohydrate esterases had increased expression in the post-treated biofilms and in samples without sugar cycles, while glucosyltransferases were highly expressed in the presence of sucrose cycles. Enrichment for functions related to nitrogen compound metabolism and organic cyclic component metabolism in groups without sucrose compared to the sucrose-treated group. Pre-treatment of the roots with cranberry reduced microbial viability and gelatinase (but not collagenase) activity (p < 0.05). SEM images showed the complexity of biofilms was maintained, with a thick extracellular polysaccharides layer.ConclusionsThis root caries model was optimized to produce complex cariogenic biofilms and root caries-like lesions, and could be used to test microbial modulation in vitro. Pre-treatments before biofilm development and cariogenic challenges were more effective than post-treatments. The clinical significance lies in the potential to apply the findings to develop varnish products for post-professional tooth prophylaxis, aiming at implementing a strategy for dysbiosis reversal in translational research.2cRgxuoMbmgt6TZPkgExXQVideo

An Experimental Protocol for the Boyden Chamber Invasion Assay With Absorbance Readout.

The phenomenon of cell invasion is an essential step in angiogenesis, embryonic development, immune responses, and cancer metastasis. In the course of cancer progression, the ability of neoplastic cells to degrade the basement membrane and penetrate neighboring tissue (or blood vessels and lymph nodes) is an early event of the metastatic cascade. The Boyden chamber assay is one of the most prevalent methods implemented to measure the pro- or anti-invasive effects of drugs, investigate signaling pathways that modulate cell invasion, and characterize the role of extracellular matrix proteins in metastasis. However, the traditional protocol of the Boyden chamber assay has some technical challenges and limitations. One such challenge is that the endpoint of the assay involves photographing and counting stained cells (in multiple fields) on porous filters. This process is very arduous, requires multiple observers, and is very time-consuming. Our improved protocol for the Boyden chamber assay involves lysis of the dye-stained cells and reading the absorbance using an ELISA reader to mitigate this challenge. We believe that our improved Boyden chamber methodology offers a standardized, high-throughput format to evaluate the efficacy of various drugs and test compounds in influencing cellular invasion in normal and diseased states. We believe that our protocol will be useful for researchers working in the fields of immunology, vascular biology, drug discovery, cancer biology, and developmental biology. Key features • Measurement of tumor invasion using human cancer cells. • Ability to measure the pro-invasive/anti-invasive activity of small molecules and biological modifiers. • Measurement of chemotaxis, chemokines, trafficking of immune cells, and proteolytic activity of matrix metalloproteinases, lysosomal hydrolysates, collagenases, and plasminogen activators in physiological and pathological conditions. • Investigation of the role of extracellular matrix proteins in the crosstalk between endothelial, epithelial, muscle, or neuronal cells and their adjacent stroma.

Apo-Lactoferrin Inhibits the Proteolytic Activity of the 110 kDa Zn Metalloprotease Produced by Mannheimia haemolytica A2.

Mannheimia haemolytica is the main etiological bacterial agent in ruminant respiratory disease. M. haemolytica secretes leukotoxin, lipopolysaccharides, and proteases, which may be targeted to treat infections. We recently reported the purification and in vivo detection of a 110 kDa Zn metalloprotease with collagenase activity (110-Mh metalloprotease) in a sheep with mannheimiosis, and this protease may be an important virulence factor. Due to the increase in the number of multidrug-resistant strains of M. haemolytica, new alternatives to antibiotics are being explored; one option is lactoferrin (Lf), which is a multifunctional iron-binding glycoprotein from the innate immune system of mammals. Bovine apo-lactoferrin (apo-bLf) possesses many properties, and its bactericidal and bacteriostatic effects have been highlighted. The present study was conducted to investigate whether apo-bLf inhibits the secretion and proteolytic activity of the 110-Mh metalloprotease. This enzyme was purified and sublethal doses of apo-bLf were added to cultures of M. haemolytica or co-incubated with the 110-Mh metalloprotease. The collagenase activity was evaluated using zymography and azocoll assays. Our results showed that apo-bLf inhibited the secretion and activity of the 110-Mh metalloprotease. Molecular docking and overlay assays showed that apo-bLf bound near the active site of the 110-Mh metalloprotease, which affected its enzymatic activity.